Treatment of pompe&#39;s disease

ABSTRACT

The invention provides methods of treating Pompe&#39;s disease using human acid alpha glucosidase. A preferred treatment regime comprises administering greater than 10 mg/kg body weight per week to a patient.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The present application derives priority from U.S. Ser. No.60/111291 filed Dec. 7, 1998, which is incorporated by reference in itsentirety for all puposes. The present application is related to U.S.Ser. No. 08/700,760 filed Jul. 29, 1996, which derives priority fromU.S. Ser. No. 60/001,796, filed Aug. 2, 1995, both of which areincorporated by reference in their entirety for all purposes.

TECHNICAL FIELD

[0002] The present invention resides in the fields of recombinantgenetics, and medicine, and is directed to enzyme-replacement therapy ofpatients with Pompe's disease.

BACKGROUND OF THE INVENTION

[0003] Like other secretory proteins, lysosomal proteins are synthesizedin the endoplasmic reticulum and transported to the Golgi apparatus.However, unlike most other secretory proteins, the lysosomal proteinsare not destined for secretion into extracellular fluids but into anintracellular organelle. Within the Golgi, lysosomal proteins undergospecial processing to equip them to reach their intracellulardestination. Almost all lysosomal proteins undergo a variety ofposttranslational modifications, including glycosylation andphosphorylation via the 6′ position of a terminal mannose group. Thephosphorylated mannose residues are recognized by specific receptors onthe inner surface of the Trans Golgi Network. The lysosomal proteinsbind via these receptors, and are thereby separated from other secretoryproteins. Subsequently, small transport vesicles containing thereceptor-bound proteins are pinched off from the Trans Golgi Network andare targeted to their intracellular destination. See generally Kornfeld,Biochem. Soc. Trans. 18, 367-374 (1990).

[0004] There are over thirty lysosomal diseases, each resulting from adeficiency of a particular lysosomal protein, usually as a result ofgenetic mutation. See, e.g., Cotran et al., Robbins Pathologic Basis ofDisease (4th ed. 1989) (incorporated by reference in its entirety forall purposes). The deficiency in the lysosomal protein usually resultsin harmful accumulation of a metabolite. For example, in Hurler's,Hunter's, Morquio's, and Sanfilippo's syndromes, there is anaccumulation of mucopolysaccharides; in Tay-Sachs, Gaucher, Krabbe,Niemann-Pick, and Fabry syndromes, there is an accumulation ofsphingolipids; and in fucosidosis and mannosidosis, there is anaccumulation of fucose-containing sphingolipids and glycoproteinfragments, and of mannose-containing oligosaccharides, respectively.

[0005] Glycogen storage disease type II (GSD II; Pompe disease; acidmaltase deficiency) is caused by deficiency of the lysosomal enzyme acidα-glucosidase (acid maltase). Two clinical forms are distinguished:early onset infantile and late onset, juvenile and adult. Infantile GSDII has its onset shortly after birth and presents with progressivemuscular weakness and cardiac failure. This clinical variantis usuallyfatal within the first two years of life. Symptoms in the late onset inadult and juvenile patients occur later in life, and only skeletalmuscles are involved. The patients eventually die due to respiratoryinsufficiency. Patients may exceptionally survive for more than sixdecades. There is a good correlation between the severity of the diseaseand the residual acid α-glucosidase activity, the activity being 10-20%of normal in late onset and less than 2% in early onset forms of thedisease (see Hirschhorn, The Metabolic and Molecular Bases of InheritedDisease (Scriver et al., eds., 7th ed., McGraw-Hill, 1995), pp.2443-2464).

[0006] Since the discovery of lysosomal enzyme deficiencies as theprimary cause of lysosomal storage diseases (see, e.g., Hers, Biochem.J. 86, 11-16 (1963)), attempts have been made to treat patients havinglysosomal storage diseases by (intravenous) administration of themissing enzyme, i.e., enzyme therapy. These experiments with enzymereplacement therapy for Pompe's disease were not successful. Eithernon-human α-glucosidase from Aspergillus niger, giving immunologicalreactions, or a form of the enzyme that is not efficiently taken up bycells (the low uptake form, mature enzyme from human placenta; seebelow) was used. Moreover, both the duration of treatment, and/or theamount of enzyme administered were insufficient (3-5). Production oflysosomal enzymes from natural sources such as human urine and bovinetestis is in theory possible, but gives low yields, and the enzymepurified is not necessarily in a form that can be taken up by tissues ofa recipient patient.

[0007] Notwithstanding the above uncertainties and difficulties, theinvention provides methods of treating patients for Pompe's diseaseusing human acid alpha glucosidase.

SUMMARY OF THE CLAIMED INVENTION

[0008] In one aspect, the invention provides methods of treating apatient with Pompe's disease. Such methods entail administering to thepatient a therapeutically effective amount of human acid alphaglucosidase. The dosage is preferably at least 10 mg/kg body weight perweek. In some methods, the dosage is at least 60 mg/kg body weight perweek or at least 120 mg/kg body weight per week. In some methods, suchdosages are administered on a single occasion per week and in othermethods on three occasions per week. In some methods, the treatment iscontainued for ate least 24 weeks. Adminstration is preferablyintravenous. The human acid alpha glucosidase is preferably obtained inthe milk of a nonhuman transgenic mammal, and is preferably predominatlyin a 110 kD form.

[0009] The methods can be used for treating patients with infantile,juvenile or adult Pompe's disease. In some methods of treating infantilePompe's disease efficacy is indicated by a patient surviving to be atleast one year old.

[0010] In some methods, levels of human acid alpha glucosidase aremonitored in the recuouebt patient. Optionally, a second dosage of humanacid alpha glucosidase can be administered if the level ofalpha-glucosidase falls below a threshold value in the patient.

[0011] In some emthods, the human alpha glucosidase is administeredintravenously and the rate of administration increases during the periodof administration. In some methods, the rate of administration increasesby at least a factor of ten during the period of administration. In somemethods, the rate of administration increases by at least a factor often within a period of five hours. In some methods, the patient isadministered a series of at least four dosages, each dosage at a higherstrength than the previous dosage. In some methods, the dosages are afirst dosage of 0.03-3 mg/kg/hr, a second dosage of 0.3-12 mg/kg/hr, athird dosage of 1-30 mg/kg/hr and a fourth dosage of 2-60 mg/kg/hr. Insome methods, the dosages are a first dosage of 0.1-1 mg/kg/hr, a seconddosage of 1-4 mg/kg/hr, a third dosage of 3-10 mg/kg/hr and a fourthdosage of 6-20 mg/kg/hr. In some methods, the dosages are a first dosageof 0.25-4 mg/kg/hr, a second dosage of 0.9-1.4 mg/kg/hr, a third dosageof 3.6-5.7 mg/kg/hr and a fourth dosage of 7.2-11.3 mg/kg/hr. In somemethods, the dosages are a first dosage of 0.3 mg/kg/hr, a second dosageof 1 mg/kg/hr, a third dosage of 4 mg/kg/hr and a fourth dosage of 12mg/kg/hr. In some methods, the first, second, third and fourth dosagesare each administered for periods of 15 min to 8 hours.

[0012] In some methods, the first, second, third and fourth dosages areadministered for periods of 1 hr, 1 hr, 0.5 hr and 3 hr respectively.

[0013] In another aspect, the invention provides a pharmaceuticalcomposition comprising human acid alpha glucosidase, human serumalbumin, and a sugar in a physiologically acceptable buffer in sterileform. Some such compositions comprise human acid alpha glucosidase,human serum albumin, and glucose in sodium phosphate buffer. Somecompositions comprise alpha glucosidase, mannitol and sucrose in anaqueous solution. In some compositions, the sugar comprises mannitol andsucrose and the concentration of mannitol is 1-3% w/w of the aqueoussolution and the concentration of sucrose is 0.1 to 1% w/w of theaqueous solution. In some compositions, the concentration of mannitol is2% w/w and the concentration of sucrose is 0.5% w/w.

[0014] The invention further provides a lyophilized composition producedby lyophilizing a pharmaceutical composition comprising human acidglucosidase, mannitol and sucrose in aqueous solution. Such acomposition can be prepared by lyophilizing a first compositioncomprising human acid alpha-glucosidase, mannitol, sucrose and anaqueous solution to produce a second composition; and reconstituting thelyophilized composition in saline to produce a third composition. Insome such compositions, the the human acid alpha-glucosidase is at 5mg/ml in both the first and third composition, the mannitol is at 2mg/ml in the first composition, the sucrose is at 0.5 mg/ml in the firstcomposition, and the saline used in the reconstituting step is 0.9% w/w.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1: A transgene containing acid α-glucosidase cDNA. Theαs1-casein exons are represented by open boxes; α-glucosidase cDNA isrepresented by a shaded box. The αs1-casein intron and flankingsequences are represented by a thick line. A thin line represents theIgG acceptor site. The transcription initiation site is marked (1^(→)),the translation initiation site (ATG), the stop codon (TAG) and thepolyadenylation site (pA).

[0016]FIG. 2 (panels A, B, C): Three transgenes containing acidα-glucosidase genomic DNA. Dark shaded areas are αs1 casein sequences,open boxes represent acids α-glucosidase exons, and the thin linebetween the open boxes represents α-glucosidase introns. Other symbolsare the same as in FIG. 1.

[0017]FIG. 3 (panels A, B, C): Construction of genomic transgenes. Theα-glucosidase exons are represented by open boxes; the α-glucosidaseintrons and nontranslated sequences are indicated by thin lines. ThepKUN vector sequences are represented by thick lines.

[0018]FIG. 4. Detection of acid α-glucosidase in milk of transgenic miceby Western blotting.

DEFINITIONS

[0019] The term “substantial identity” or “substantial homology” meansthat two peptide sequences, when optimally aligned, such as by theprograms GAP or BESTFIT using default gap weights, share at least 65percent sequence identity, preferably at least 80 or 90 percent sequenceidentity, more preferably at least 95 percent sequence identity or more(e.g., 99 percent sequence identity). Preferably, residue positionswhich are not identical differ by conservative amino acid substitutions.

[0020] The term “substantially pure” or “isolated” means an objectspecies has been identified and separated and/or recovered from acomponent of its natural environment. Usually, the object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 to 90 percent by weight ofall macromolecular species present in the composition. Most preferably,the object species is purified to essential homogeneity (contaminantspecies cannot be detected in the composition by conventional detectionmethods) wherein the composition consists essentially of derivatives ofa single macromolecular species.

[0021] A DNA segment is operably linked when placed into a functionalrelationship with another DNA segment. For example, DNA for a signalsequence is operably linked to DNA encoding a polypeptide if it isexpressed as a preprotein that participates in the secretion of thepolypeptide; a promoter or enhancer is operably linked to a codingsequence if it stimulates the transcription of the sequence. Generally,DNA sequences that are operably linked are contiguous, and in the caseof a signal sequence both contiguous and in reading phase. However,enhancers need not be contiguous with the coding sequences whosetranscription they control. Linking is accomplished by ligation atconvenient restriction sites or at adapters or linkers inserted in lieuthereof.

[0022] An exogenous DNA segment is one foreign to the cell, orhomologous to a DNA segment of the cell but in an unnatural position inthe host cell genome. Exogenous DNA segments are expressed to yieldexogenous polypeptides.

[0023] In a transgenic mammal, all, or substantially all, of thegermline and somatic cells contain a transgene introduced into themammal or an ancestor of the mammal at an early embryonic stage.

DETAILED DESCRIPTION

[0024] The invention provides transgenic nonhuman mammals secreting alysosomal protein into their milk. Secretion is achieved byincorporation of a transgene encoding a lysosomal protein and regulatorysequences capable of targeting expression of the gene to the mammarygland. The transgene is expressed, and the expression productposttranslationally modified within the mammary gland, and then secretedin milk. The posttranslational modification can include steps ofglycosylation and phosphorylation to produce a mannose-6 phosphatecontaining lysosomal protein.

[0025] A. Lysosomal Genes

[0026] The invention provides transgenic nonhuman mammals expressing DNAsegments containing any of the more than 30 known genes encodinglysosomal enzymes and other types of lysosomal proteins, includingα-glucosidase, α-L-iduronidase, iduronate-sulfate sulfatase,hexosaminidase A and B, ganglioside activator protein, arylsulfatase Aand B, iduronate sulfatase, heparan N-sulfatase, galacto-ceramidase,α-galactosylceramidase A, sphingomyelinase, α-fucosidase, α-mannosidase,aspartylglycosamine amide hydrolase, acid lipase,N-acetyl-α-D-glucosamine-6-sulphate sulfatase, α-and β-galactosidase,β-glucuronidase, β-mannosidase, ceramidase, galacto-cere-brosidase,α-N-acetylgalactosaminidase, and protective protein and others.Transgenic mammals expressing allelic, cognate and induced variants ofany of the known lysosomal protein gene sequences are also included.Such variants usually show substantial sequence identity at the aminoacid level with known lysosomal protein genes. Such variants usuallyhybridize to a known gene under stringent conditions or crossreact withantibodies to a polypeptide encoded by one of the known genes.

[0027] DNA clones containing the genomic or cDNA sequences of many ofthe known genes encoding lysosomal proteins are available. (Scott etal., Am. J. Hum. Genet. 47, 802-807 (1990); Wilson et al., PNAS 87,8531-8535 (1990); Stein et al., J. Biol. Chem. 264, 1252-1259 (1989);Ginns et al., Biochem. Biophys. Res. Comm. 123, 574-580 (1984);Hoefsloot et al., EMBO J. 7, 1697-1704 (1988); Hoefsloot et al.,Biochem. J. 272, 473-479 (1990); Meyerowitz & Proia, PNAS 81, 5394-5398(1984); Scriver et al., supra, part 12, pages 2427-2882 and referencescited therein)) Other examples of genomic and cDNA sequences areavailable from GenBank. To the extent that additional cloned sequencesof lysosomal genes are required, they may be obtained from genomic orcDNA libraries (preferably human) using known lysosomal protein DNAsequences or antibodies to known lysosomal proteins as probes.

[0028] B. Conformation of Lysosomal Proteins

[0029] Recombinant lysosomal proteins are preferably processed to havethe same or similar structure as naturally occurring lysosomal proteins.Lysosomal proteins are glycoproteins that are synthesized on ribosomesbound to the endoplasmic reticulum (RER). They enter this organelleco-translationally guided by an N-terminal signal peptide (Ng et al.,Current Opinion in Cell Biology 6, 510-516 (1994)). The N-linkedglycosylation process starts in the RER with the en bloc transfer of thehigh-mannose oligosaccharide precursor Glc3Man9 GlcNAc2 from a dolicholcarrier. Carbohydrate chain modification starts in the RER and continuein the Golgi apparatus with the removal of the three outermost glucoseresidues by glycosidases I and II. Phosphorylation is a two-stepprocedure in which first N-acetyl-gluco-samine-1-phosphate is coupled toselect mannose groups by a lysosomal protein specific transferase, andsecond, the N-acetyl-gluco-samine is cleaved by a diesterase (Goldberget al., Lysosomes: Their Role in Protein Breakdown (Academic Press Inc.,London, 1987), pp. 163-191). Cleavage exposes mannose 6-phosphate as arecognition marker and ligand for the mannose 6-phosphate receptormediating transport of most lysosomal proteins to the lysosomes(Kornfeld, Biochem. Soc. Trans. 18, 367-374 (1992)).

[0030] In addition to carbohydrate chain modification, most lysosomalproteins undergo proteolytic processing, in which the first event isremoval of the signal peptide. The signal peptide of most lysosomalproteins is cleaved after translocation by signal peptidase after whichthe proteins become soluble. There is suggestive evidence that thesignal peptide of acid α-glucosidase is cleaved after the enzyme hasleft the RER, but before it has entered the lysosome or the secretorypathway (Wisselaar et al., J. Biol. Chem. 268, 2223-2231 (1993)). Theproteolytic processing of acid α-glucosidase is complex and involves aseries of steps in addition to cleavage of the signal peptide takingplace at various subcellular locations. Polypeptides are cleaved off atboth the N and C terminal ends, whereby the specific catalytic activityis increased. The main species recognized are a 110/100 kD precursor, a95 kD intermediate and 76 kD and 70 kD mature forms. (Hasilik et al., J.Biol. Chem. 255, 4937-4945 (1980); Oude Elferink et al., Eur. J.Biochem. 139, 489-495 (1984); Reuser et al., J. Biol. Chem. 260,8336-8341 (1985); Hoefsloot et al., EMBO J. 7, 1697-1704 (1988)). Thepost translational processing of natural human acid α-glucosidase and ofrecombinant forms of human acid α-glucosidase as expressed in culturedmammalian cells like COS cells, BHK cells and CHO cells is similar(Hoefsloot et al., (1990) supra; Wisselaar et al., (1993) supra.

[0031] Authentic processing to generate lysosomal proteinsphosphorylated at the 6′ position of the mannose group can be tested bymeasuring uptake of a substrate by cells bearing a receptor for mannose6-phosphate. Correctly modified substrates are taken up faster thanunmodified substrates, and in a manner whereby uptake of the modifiedsubstrate can be competitively inhibited by addition of mannose6-phosphate.

[0032] C. Transgene Design

[0033] Transgenes are designed to target expression of a recombinantlysosomal protein to the mammary gland of a transgenic nonhuman mammalharboring the transgene. The basic approach entails operably linking anexogenous DNA segment encoding the protein with a signal sequence, apromoter and an enhancer. The DNA segment can be genomic, minigene(genomic with one or more introns omitted), cDNA, a YAC fragment, achimera of two different lysosomal protein genes, or a hybrid of any ofthese. Inclusion of genomic sequences generally leads to higher levelsof expression. Very high levels of expression might overload thecapacity of the mammary gland to perform posttranslation modifications,and secretion of lysosomal proteins. However, the data presented belowindicate that substantial posttranslational modification occursincluding the formation of mannose 6-phosphate groups, notwithstanding ahigh expression level in the mg/ml range. Substantial modification meansthat at least about 10, 25, 50, 75 or 90% of secreted molecules bear atleast one mannose 6-phosphate group. Thus, genomic constructs or hybridcDNA-genomic constructs are generally preferred.

[0034] In genomic constructs, it is not necessary to retain all intronicsequences. For example, some intronic sequences can be removed to obtaina smaller transgene facilitating DNA manipulations and subsequentmicroinjection. See Archibald et al., WO 90/05188 (incorporated byreference in its entirety for all purposes). Removal of some introns isalso useful in some instances to reduce expression levels and therebyensure that posttranslational modification is substantially complete. Inother instances excluding an intron such as intron one from the genomicsequence of acid α-glucosidase leads to a higher expression of themature enzyme. It is also possible to delete some or all of noncodingexons. In some transgenes, selected nucleotides in lysosomal proteinencoding sequences are mutated to remove proteolytic cleavage sites.

[0035] Because the intended use of lysosomal proteins produced bytransgenic mammals is usually administration to humans, the species fromwhich the DNA segment encoding a lysosomal protein sequence is obtainedis preferably human. Analogously if the intended use were in veterinarytherapy (e.g., on a horse, dog or cat), it is preferable that the DNAsegment be from the same species.

[0036] The promoter and enhancer are from a gene that is exclusively orat least preferentially expressed in the mammary gland (i.e., amammary-gland specific gene). Preferred genes as a source of promoterand enhancer include β-casein, κ-casein, αS1-casein, αS2-casein,β-lactoglobulin, whey acid protein, and α-lactalbumin. The promoter andenhancer are usually but not always obtained from the same mammary-glandspecific gene. This gene is sometimes but not necessarily from the samespecies of mammal as the mammal into which the transgene is to beexpressed. Expression regulation sequences from other species such asthose from human genes can also be used. The signal sequence must becapable of directing the secretion of the lysosomal protein from themammary gland. Suitable signal sequences can be derived from mammaliangenes encoding a secreted protein. Surprisingly, the natural signalsequences of lysosomal proteins are suitable, notwithstanding that theseproteins are normally not secreted but targeted to an intracellularorganelle. In addition to such signal sequences, preferred sources ofsignal sequences are the signal sequence from the same gene as thepromoter and enhancer are obtained. Optionally, additional regulatorysequences are included in the transgene to optimize expression levels.Such sequences include 5′ flanking regions, 5′ transcribed butuntranslated regions, intronic sequences, 3′ transcribed butuntranslated regions, polyadenylation sites, and 3′ flanking regions.Such sequences are usually obtained either from the mammary-glandspecific gene from which the promoter and enhancer are obtained or fromthe lysosomal protein gene being expressed. Inclusion of such sequencesproduces a genetic milieu simulating that of an authentic mammary glandspecific gene and/or that of an authentic lysosomal protein gene. Thisgenetic milieu results in some cases (e.g., bovine αS1-casein) in higherexpression of the transcribed gene. Alternatively, 3′ flanking regionsand untranslated regions are obtained from other heterologous genes suchas the β-globin gene or viral genes. The inclusion of 3′ and 5′untranslated regions from a lysosomal protein gene, or otherheterologous gene can also increase the stability of the transcript.

[0037] In some embodiments, about 0.5, 1, 5, 10, 15, 20 or 30 kb of 5′flanking sequence is included from a mammary specific gene incombination with about 1, 5, 10, 15, 20 or 30 kb or 3′ flanking sequencefrom the lysosomal protein gene being expressed. If the protein isexpressed from a cDNA sequence, it is advantageous to include anintronic sequence between the promoter and the coding sequence. Theintronic sequence is preferably a hybrid sequence formed from a 5′portion from an intervening sequence from the first intron of themammary gland specific region from which the promoter is obtained and a3′ portion from an intervening sequence of an IgG intervening sequenceor lysosomal protein gene. See DeBoer et al., WO 91/08216 (incorporatedby reference in its entirety for all purposes).

[0038] A preferred transgene for expressing a lysosomal proteincomprises a cDNA-genomic hybrid lysosomal protein gene linked 5′ to acasein promoter and enhancer. The hybrid gene includes the signalsequence, coding region, and a 3′ flanking region from the lysosomalprotein gene. Optionally, the cDNA segment includes an intronic sequencebetween the 5′ casein and untranslated region of the gene encoding thelysosomal protein. Of course, corresponding cDNA and genomic segmentscan also be fused at other locations within the gene provided acontiguous protein can be expressed from the resulting fusion.

[0039] Other preferred transgenes have a genomic lysosomal proteinsegment linked 5′ to casein regulatory sequences. The genomic segment isusually contiguous from the 5′ untranslated region to the 3′ flankingregion of the gene. Thus, the genomic segment includes a portion of thelysosomal protein 5′ untranslated sequence, the signal sequence,alternating introns and coding exons, a 3′ untranslated region, and a 3′flanking region. The genomic segment is linked via the 5′ untranslatedregion to a casein fragment comprising a promoter and enhancer andusually a 5′ untranslated region.

[0040] DNA sequence information is available for all of the mammarygland specific genes listed above, in at least one, and often severalorganisms. See, e.g., Richards et al., J. Biol. Chem. 256, 526-532(1981) (α-lactalbumin rat); Campbell et al., Nucleic Acids Res. 12,8685-8697 (1984) (rat WAP); Jones et al., J. Biol. Chem. 260, 7042-7050(1985)) (rat β-casein); Yu-Lee & Rosen, J. Biol. Chem. 258, 10794-10804(1983) (rat γ-casein)); Hall, Biochem. J. 242, 735-742 (1987)(α-lactalbumin human); Stewart, Nucleic Acids Res. 12, 389 (1984)(bovine αs1 and K casein cDNAs); Gorodetsky et al., Gene 66, 87-96(1988) (bovine β casein); Alexander et al., Eur. J. Biochem. 178,395-401 (1988) (bovine K casein); Brignon et al., FEBS Lett. 188, 48-55(1977) (bovine αS2 casein); Jamieson et al., Gene 61, 85-90 (1987),Ivanov et al., Biol. Chem. Hoppe-Seyler 369, 425-429 (1988), Alexanderet al., Nucleic Acids Res. 17, 6739 (1989) (bovine β lactoglobulin);Vilotte et al., Biochimie 69, 609-620 (1987) (bovine α-lactalbumin)(incorporated by reference in their entirety for all purposes). Thestructure and function of the various milk protein genes are reviewed byMercier & Vilotte, J. Dairy Sci. 76, 3079-3098 (1993) (incorporated byreference in its entirety for all purposes). To the extent thatadditional sequence data might be required, sequences flanking theregions already obtained could be readily cloned using the existingsequences as probes. Mammary-gland specific regulatory sequences fromdifferent organisms are likewise obtained by screening libraries fromsuch organisms using known cognate nucleotide sequences, or antibodiesto cognate proteins as probes.

[0041] General strategies and exemplary transgenes employing αS1-caseinregulatory sequences for targeting the expression of a recombinantprotein to the mammary gland are described in more detail in DeBoer etal., WO 91/08216 and WO 93/25567 (incorporated by reference in theirentirety for all purposes). Examples of transgenes employing regulatorysequences from other mammary gland specific genes have also beendescribed. See, e.g., Simon et al., Bio/Technology 6, 179-183 (1988) andWO88/00239 (1988) (β-lactoglobulin regulatory sequence for expression insheep); Rosen, EP 279,582 and Lee et al., Nucleic Acids Res. 16,1027-1041 (1988) (β-casein regulatory sequence for expression in mice);Gordon, Biotechnology 5, 1183 (1987) (WAP regulatory sequence forexpression in mice); WO 88/01648 (1988) and Eur. J. Biochem. 186, 43-48(1989) (α-lactalbumin regulatory sequence for expression in mice)(incorporated by reference in their entirety for all purposes).

[0042] The expression of lysosomal proteins in the milk from transgenescan be influenced by co-expression or functional inactivation (i.e.,knock-out) of genes involved in post translational modification andtargeting of the lysosomal proteins. The data in the Examples indicatethat surprisingly mammary glands already express modifying enzymes atsufficient quantities to obtain assembly and secretion of mannose6-phosphate containing proteins at high levels. However, in sometransgenic mammals expressing these proteins at high levels, it issometimes preferable to supplement endogenous levels of processingenzymes with additional enzyme resulting from transgene expression. Suchtransgenes are constructed employing similar principles to thosediscussed above with the processing enzyme coding sequence replacing thelysosomal protein coding sequence in the transgene. It is not generallynecessary that posttranslational processing enzymes be secreted. Thus,the secretion signal sequence linked to the lysosomal protein codingsequence is replaced with a signal sequence that targets the processingenzyme to the endoplasmic reticulum without secretion. For example, thesignal sequences naturally associated with these enzymes are suitable.

[0043] D. Transgenesis

[0044] The transgenes described above are introduced into nonhumanmammals. Most nonhuman mammals, including rodents such as mice and rats,rabbits, ovines such as sheep and goats, porcines such as pigs, andbovines such as cattle and buffalo, are suitable. Bovines offer anadvantage of large yields of milk, whereas mice offer advantages of easeof transgenesis and breeding. Rabbits offer a compromise of theseadvantages. A rabbit can yield 100 ml milk per day with a proteincontent of about 14% (see Buhler et al., Bio/Technology 8, 140 (1990))(incorporated by reference in its entirety for all purposes), and yetcan be manipulated and bred using the same principles and with similarfacility as mice. Nonviviparous mammals such as a spiny anteater orduckbill platypus are typically not employed.

[0045] In some methods of transgenesis, transgenes are introduced intothe pronuclei of fertilized oocytes. For some animals, such as mice andrabbits, fertilization is performed in vivo and fertilized ova aresurgically removed. In other animals, particularly bovines, it ispreferable to remove ova from live or slaughterhouse animals andfertilize the ova in vitro. See DeBoer et al., WO 91/08216. In vitrofertilization permits a transgene to be introduced into substantiallysynchronous cells at an optimal phase of the cell cycle for integration(not later than S-phase). Transgenes are usually introduced bymicroinjection. See U.S. Pat. No. 4,873,292. Fertilized oocytes are thencultured in vitro until a pre-implantation embryo is obtained containingabout 16-150 cells. The 16-32 cell stage of an embryo is described as amorula. Pre-implantation embryos containing more than 32 cells aretermed blastocysts. These embryos show the development of a blastocoelecavity, typically at the 64 cell stage. Methods for culturing fertilizedoocytes to the pre-implantation stage are described by Gordon et al.,Methods Enzymol. 101, 414 (1984); Hogan et al., Manipulation of theMouse Embryo: A Laboratory Manual, C.S.H.L. New York (1986) (mouseembryo); and Hammer et al., Nature 315, 680 (1985) (rabbit and porcineembryos); Gandolfi et al. J. Reprod. Fert. 81, 23-28 (1987); Rexroad etal., J. Anim. Sci. 66, 947-953 (1988) (ovine embryos) and Eyestone etal. J. Reprod. Fert. 85, 715-720 (1989); Camous et al., J. Reprod. Fert.72, 779-785 (1984); and Heyman et al. Theriogenology 27, 5968 (1987)(bovine embryos) (incorporated by reference in their entirety for allpurposes). Sometimes pre-implantation embryos are stored frozen for aperiod pending implantation. Pre-implantation embryos are transferred tothe oviduct of a pseudopregnant female resulting in the birth of atransgenic or chimeric animal depending upon the stage of developmentwhen the transgene is integrated. Chimeric mammals can be bred to formtrue germline transgenic animals.

[0046] Alternatively, transgenes can be introduced into embryonic stemcells (ES). These cells are obtained from preimplantation embryoscultured in vitro. Bradley et al., Nature 309, 255-258 (1984)(incorporated by reference in its entirety for all purposes). Transgenescan be introduced into such cells by electroporation or microinjection.Transformed ES cells are combined with blastocysts from a non-humananimal. The ES cells colonize the embryo and in some embryos form thegermline of the resulting chimeric animal. See Jaenisch, Science, 240,1468-1474 (1988) (incorporated by reference in its entirety for allpurposes). Alternatively, ES cells can be used as a source of nuclei fortransplantation into an enucleated fertilized oocyte giving rise to atransgenic mammal.

[0047] For production of transgenic animals containing two or moretransgenes, the transgenes can be introduced simultaneously using thesame procedure as for a single transgene. Alternatively, the transgenescan be initially introduced into separate animals and then combined intothe same genome by breeding the animals. Alternatively, a firsttransgenic animal is produced containing one of the transgenes. A secondtransgene is then introduced into fertilized ova or embryonic stem cellsfrom that animal. In some embodiments, transgenes whose length wouldotherwise exceed about 50 kb, are constructed as overlapping fragments.Such overlapping fragments are introduced into a fertilized oocyte orembryonic stem cell simultaneously and undergo homologous recombinationin vivo. See Kay et al., WO 92/03917 (incorporated by reference in itsentirety for all purposes).

[0048] E. Characteristics of Transgenic Mammals

[0049] Transgenic mammals of the invention incorporate at least onetransgene in their genome as described above. The transgene targetsexpression of a DNA segment encoding a lysosomal protein at leastpredominantly to the mammary gland. Surprisingly, the mammary glands arecapable of expressing proteins required for authentic posttranslationprocessing including steps of oligosaccharide addition andphosphorylation. Processing by enzymes in the mammary gland results inphosphorylation of the 6′ position of mannose groups.

[0050] Lysosomal proteins can be secreted at high levels of at least 10,50, 100, 500, 1000, 2000, 5000 or 10,000 μg/ml. Surprisingly, thetransgenic mammals of the invention exhibit substantially normal health.Secondary expression of lysosomal proteins in tissues other than themammary gland does not occur to an extent sufficient to causedeleterious effects. Moreover, exogenous lysosomal protein produced inthe mammary gland is secreted with sufficient efficiency that nosignificant problem is presented by deposits clogging the secretoryapparatus.

[0051] The age at which transgenic mammals can begin producing milk, ofcourse, varies with the nature of the animal. For transgenic bovines,the age is about two-and-a-half years naturally or six months withhormonal stimulation, whereas for transgenic mice the age is about 5-6weeks. Of course, only the female members of a species are useful forproducing milk. However, transgenic males are also of value for breedingfemale descendants. The sperm from transgenic males can be stored frozenfor subsequent in vitro fertilization and generation of femaleoffspring.

[0052] F. Recovery of Proteins from Milk

[0053] Transgenic adult female mammals produce milk containing highconcentrations of exogenous lysosomal protein. The protein can bepurified from milk, if desired, by virtue of its distinguishing physicaland chemical properties, and standard purification procedures such asprecipitation, ion exchange, molecular exclusion or affinitychromatography. See generally Scopes, Protein Purification(Springer-Verlag, New York, 1982).

[0054] Purification of human acid α-glucosidase from milk can be carriedout by defatting of the transgenic milk by centrifugation and removal ofthe fat, followed by removal of caseins by high speed centrifugationfollowed by dead-end filtration (i.e., dead-end filtration by usingsuccessively declining filter sizes) or cross-flow filtration, or;removal of caseins directly by cross-flow filtration. Human acidα-glucosidase is purified by chromatography, including Q Sepharose FF(or other anion-exchange matrix), hydrophobic interaction chromatography(HIC), metal-chelating Sepharose, or lectins coupled to Sepharose (orother matrices).

[0055] Q Sepharose Fast Flow chromatography may be used to purify humanacid α-glucosidase present in filtered whey or whey fraction as follows:a Q Sepharose Fast Flow (QFF; Pharmacia) chromatography (Pharmacia XK-50column, 15 cm bed height; 250 cm/hr flow rate) the column wasequilibrated in 20 mM sodiumphosphate buffer, pH 7.0 (buffer A); theS/D-incubated whey fraction (about 500 to 600 ml) is loaded and thecolumn is washed with 4-6 column volumes (cv) of buffer A (20 mM sodiumphosphate buffer, pH 7.0). The human acid α-glucosidase fraction iseluted from the Q FF column with 2-3 cv buffer A, containing 100 mMNaCl.

[0056] The Q FF Sepharose human acid α-glucosidase containing fractioncan be further purified using Phenyl Sepharose High Performancechromatography. For example, 1 vol. of 1M ammonium sulphate is added tothe Q FF Sepharose human acid α-glucosidase eluate while stirringcontinuously. Phenyl HP (Pharmacia) column chromatography (PharmaciaXK-50 column, 15 cm bed height; 150 cm/hr flow rate) is then done atroom temperature by equilibrating the column in 0.5 M ammonium sulphate,50 mM sodiumphosphate buffer pH 6.0 (buffer C), loading the 0.5 Mammoniumsulphate-incubated human acid α-glucosidase eluate (from Q FFSepharose), washing the column with 2-4 cv of buffer C, and eluting thehuman acid α-glucosidase was eluted from the Phenyl HP column with 3-5cv buffer D (50 mM sodiumphosphate buffer at pH 6.0). Alternativemethods and additional methods for further purifying human acidα-glucosidase will be apparent to those of skill. For example, seeUnited Kingdom patent application 998 07464.4 (incorporated by referencein its entirety for all purposes).

[0057] G. Uses of Recombinant Lysosomal Proteins

[0058] The recombinant lysosomal proteins produced according to theinvention find use in enzyme replacement therapeutic procedures. Apatient having a genetic or other deficiency resulting in aninsufficiency of functional lysosomal enzyme can be treated byadministering exogenous enzyme to the patient. Patients in need of suchtreatment can be identified from symptoms (e.g., Hurler's syndromesymptoms include Dwarfism, corneal clouding, hepatosplenomegaly,valvular lesions, coronary artery lesions, skeletal deformities, jointstiffness and progressive mental retardation). Alternatively, oradditionally, patients can be diagnosed from biochemical analysis of atissue sample to reveal excessive accumulation of a characteristicmetabolite processed by a particular lysosomal enzyme or by enzyme assayusing an artificial or natural substrate to reveal deficiency of aparticular lysosomal enzyme activity. For most diseases, diagnosis canbe made by measuring the particular enzyme deficiency or by DNA analysisbefore occurrence of symptoms or excessive accumulation of metabolites(Scriver et al., supra, chapters on lysosomal storage disorders). All ofthe lysosomal storage diseases are hereditary. Thus, in offspring fromfamilies known to have members suffering from lysosomal diseases, it issometimes advisable to commence prophylactic treatment even before adefinitive diagnosis can be made.

[0059] Pharmaceutical Compositions

[0060] In some methods, lysosomal enzymes are administered in purifiedform together with a pharmaceutical carrier as a pharmaceuticalcomposition. The preferred form depends on the intended mode ofadministration and therapeutic application. The pharmaceutical carriercan be any compatible, nontoxic substance suitable to deliver thepolypeptides to the patient. Sterile water, alcohol, fats, waxes, andinert solids may be used as the carrier. Pharmaceutically-acceptableadjuvants, buffering agents, dispersing agents, and the like, may alsobe incorporated into the pharmaceutical compositions.

[0061] The concentration of the enzyme in the pharmaceutical compositioncan vary widely, i.e., from less than about 0.1% by weight, usuallybeing at least about 1% by weight to as much as 20% by weight or more.

[0062] For oral administration, the active ingredient can beadministered in solid dosage forms, such as capsules, tablets, andpowders, or in liquid dosage forms, such as elixirs, syrups, andsuspensions. Active component(s) can be encapsulated in gelatin capsulestogether with inactive ingredients and powdered carriers, such asglucose, lactose, sucrose, mannitol, starch, cellulose or cellulosederivatives, magnesium stearate, stearic acid, sodium saccharin, talcum,magnesium carbonate and the like. Examples of additional inactiveingredients that may be added to provide desirable color, taste,stability, buffering capacity, dispersion or other known desirablefeatures are red iron oxide, silica gel, sodium lauryl sulfate, titaniumdioxide, edible white ink and the like. Similar diluents can be used tomake compressed tablets. Both tablets and capsules can be manufacturedas sustained release products to provide for continuous release ofmedication over a period of hours. Compressed tablets can be sugarcoated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric-coated for selectivedisintegration in the gastrointestinal tract. Liquid dosage forms fororal administration can contain coloring and flavoring to increasepatient acceptance.

[0063] A typical composition for intravenous infusion could be made upto contain 100 to 500 ml of sterile 0.9% NaCl or 5% glucose optionallysupplemented with a 20% albumin solution and 100 to 500 mg of an enzyme.A typical pharmaceutical compositions for intramuscular injection wouldbe made up to contain, for example, 1 ml of sterile buffered water and 1to 10 mg of the purified alpha glucosidase of the present invention.Methods for preparing parenterally administrable compositions are wellknown in the art and described in more detail in various sources,including, for example, Remington's Pharmaceutical Science (15th ed.,Mack Publishing, Easton, Pa., 1980) (incorporated by reference in itsentirety for all purposes).

[0064] AGLU can be formulated in 10 mM sodium phosphate buffer pH 7.0.Small amounts of ammonium sulphate are optionally present (<10 mM). Theenzyme is typically kept frozen at about −70° C., and thawed before use.Alternatively, the enzyme may be stored cold (e.g., at about 4° C. to 8°C.) in solution. In some embodiments, AGLU solutions comprise a buffer(e.g., sodium phosphate, potassium phosphate or other physiologicallyacceptable buffers), a simple carbohydrate (e.g., sucrose, glucose,maltose, mannitol or the like), proteins (e.g., human serum albumin),and/or surfactants (e.g., polysorbate 80 (Tween-80), cremophore-EL,cremophore-R, labrofil, and the like).

[0065] AGLU can also be stored in lyophilized form. For lyophilization,AGLU can be formulated in a solution containing mannitol, and sucrose ina phosphate buffer. The concentration of sucrose should be sufficient toprevent aggregation of AGLU on reconstitution. The concentration ofmannitol should be sufficient to significantly reduce the time otherwiseneeded for lyophilization. The concentrations of mannitol and sucroseshould, however, be insufficient to cause unacceptable hypertonicity onreconstitution. Concentrations of mannitol and sucrose of 1-3 mg/ml and0.1-1.0 mg/ml respectively are suitable. Preferred concentrations are 2mg/ml mannitol and 0.5 mg/ml sucrose. AGLU is preferably at 5 mg/mlbefore lyophilization and after reconstitution. Saline preferably at0.9% is a preferred solution for reconstitution.

[0066] For AGLU purified from rabbit milk, a small amount of impurities(e.g., up to about 5%) can be tolerated. Possible impurities may bepresent in the form of rabbit whey proteins. Other possible impuritiesare structural analogues (e.g., oligomers and aggregates) andtruncations of AGLU. Current batches indicate that the AGLU produced intransgenic rabbits is >95% pure. The largest impurities are rabbit wheyproteins, although on gel electrophoresis, AGLU bands of differingmolecular weights are also seen.

[0067] Infusion solutions should be prepared aseptically in a laminarair flow hood. The appropriate amount of AGLU should be removed from thefreezer and thawed at room temperature. Infusion solutions can beprepared in glass infusion bottles by mixing the appropriate amount ofAGLU finished product solution with an adequate amount of a solutioncontaining human serum albumin (HSA) and glucose. The finalconcentrations can be 1% HSA and 4% glucose for 25-200 mg doses and 1%HSA and 4% glucose for 400-800 mg doses. HSA and AGLU can be filteredwith a 0.2 μm syringe filter before transfer into the infusion bottlecontaining 5% glucose. Alternatively, AGLU can be reconstituted insaline solution, preferably 0.9% for infusion. Solutions of AGLU forinfusion have been shown to be stable for up to 7 hours at roomtemperature. Therefore the AGLU solution is preferably infused withinseven hours of preparation.

[0068] Therapeutic Methods

[0069] The present invention provides effective methods of treatingPompe's disease. These methods are premised in part on the availabilityof large amounts of human acid alpha glucosidase in a form that iscatalytically active and in a form that can be taken up by tissues,particularly, liver, heart and muscle (e.g., smooth muscle, striatedmuscle, and cardiac muscle), of a patient being treated. Such human acidalpha-glucosidase is provided from e.g., the transgenic animalsdescribed in the Examples. The alpha-glucosidase is preferablypredominantly (i.e., >50%) in the precursor form of about 100-110 kD.(The apparent molecular weight or relative mobility of the 100-110 kDprecursor may vary somewhat depending on the method of analysis used,but is typically within the range 95 kD and 120 kD.) Given thesuccessful results with human acid alpha-glucosidase in the transgenicanimals discussed in the Examples, it is possible that other sources ofhuman alpha-glucosidase, such as resulting from cellular expressionsystems, can also be used. For example, an alternative way to producehuman acid α-glucosidase is to transfect the acid α-glucosidase geneinto a stable eukaryotic cell line (e.g., CHO) as a cDNA or genomicconstruct operably linked to a suitable promoter. However, it is morelaborious to produce the large amounts of human acid alpha glucosidaseneeded for clinical therapy by such an approach.

[0070] The pharmaceutical compositions of the present invention areusually administered intravenously. Intradermal, intramuscular or oraladministration is also possible in some circumstances. The compositionscan be administered for prophylactic treatment of individuals sufferingfrom, or at risk of, a lysosomal enzyme deficiency disease. Fortherapeutic applications, the pharmaceutical compositions areadministered to a patient suffering from established disease in anamount sufficient to reduce the concentration of accumulated metaboliteand/or prevent or arrest further accumulation of metabolite. Forindividuals at risk of lysosomal enzyme deficiency disease, thepharmaceutical compositions are administered prophylactically in anamount sufficient to either prevent or inhibit accumulation ofmetabolite. An amount adequate to accomplish this is defined as a“therapeutically-” or “prophylactically-effective dose.” Such effectivedosages will depend on the severity of the condition and on the generalstate of the patient's health.

[0071] In the present methods, human acid alpha glucosidase is usuallyadministered at a dosage of 10 mg/kg patient body weight or more perweek to a patient. Often dosages are greater than 10 mg/kg per week.Dosages regimes can range from 10 mg/kg per week to at least 1000 mg/kgper week. Typically dosage regimes are 10 mg/kg per week, 15 mg/kg perweek, 20 mg/kg per week, 25 mg/kg per week, 30 mg/kg per week, 35 mg/kgper week, 40 mg/kg week, 45 mg/kg per week, 60 mg/kg week, 80 mg/kg perweek and 120 mg/kg per week. In preferred regimes 10 mg/kg, 15 mg/kg, 20mg/kg, 30 mg/kg or 40 mg/kg is administered once, twice or three timesweekly. Treatment is typically continued for at least 4 weeks, sometimes24 weeks, and sometimes for the life of the patient. Treatment ispreferably administered i.v. Optionally, levels of humanalpha-glucosidase are monitored following treatment (e.g., in theplasmaor muscle) and a further dosage is administered when detected levelsfall substantially below (e.g., less than 20%) of values in normalpersons.

[0072] In some methods, human acid alpha glucosidase is administered atan initially “high” dose (i.e., a “loading dose”), followed byadministration of a lower doses (i.e., a “maintenance dose”). An exampleof a loading dose is at least about 40 mg/kg patient body weight 1 to 3times per week (e.g., for 1, 2, or 3 weeks). An example of a maintenancedose is at least about 5 to at least about 10 mg/kg patient body weightper week, or more, such as 20 mg/kg per week, 30 mg/kg per week, 40mg/kg week.

[0073] In some methods, a dosage is administered at increasing rateduring the dosage period. Such can be achieved by increasing the rate offlow intravenous infusion or by using a gradient of increasingconcentration of alpha-glucosidase administered at constant rate.Administration in this manner reduces the risk of immunogenic reaction.In some dosages, the rate of administration measured in units of alphaglucosidase per unit time increases by at least a factor of ten.Typically, the intravenous infusion occurs over a period of severalhours (e.g., 1-10 hours and preferably 2-8 hours, more preferably 3-6hours), and the rate of infusion is increased at intervals during theperiod of administration.

[0074] Suitable dosages (all in mg/kg/hr) for infusion at increasingrates are shown in table 1 below. The first column of the tableindicates periods of time in the dosing schedule. For example, thereference to 0-1 hr refers to the first hour of the dosing. The fifthcolumn of the table shows the range of doses than can be used at eachtime period. The fourth column shows a narrower included range ofpreferred dosages. The third column indicates upper and lower values ofdosages administered in an exemplary clinical trial. The second columnshows particularly preferred dosages, these representing the mean of therange shown in the third column of table 1. TABLE 1 Lower & PreferredTime Mean Doses (I) Upper Values Range Range 0-1 hr: 0.3 mg/kg/hr0.25-0.4  0.1-1   0.03-3   1-2 hr:   1 mg/kg/hr 0.9-1.4 1-4 0.3-12 2-2.5 hr:   4 mg/kg/hr 3.6-5.7  3-10  1-30 2.5-5.6 hr.  12 mg/kg/hr 7.2-11.3  6-20  2-60

[0075] The methods are effective on patients with both early onset(infantile) and late onset (juvenile and adult) Pompe's disease. Inpatients with the infantile form of Pompe's disease symptoms becomeapparent within the first 4 months of life. Mostly, poor motordevelopment and failure to thrive are noticed first. On clinicalexamination, there is generalized hypotonia with muscle wasting,increased respiration rate with sternal retractions, moderateenlargement of the liver, and protrusion of the tongue. Ultrasoundexamination of the heart shows a progressive hypertrophiccardiomyopathy, eventually leading to insufficient cardiac output. TheECG is characterized by marked left axis deviation, a short PR interval,large QRS complexes, inverted T waves and ST depression. The diseaseshows a rapidly progressive course leading to cardiorespiratory failurewithin the first year of life. On histological examination at autopsylysosomal glycogen storage is observed in various tissues, and is mostpronounced in heart and skeletal muscle. Treatment with human acid alphaglucosidase in the present methods results in a prolongation of life ofsuch patients (e.g., greater than 1, 2, 5 years up to a normallifespan). Treatment can also result in elimination or reduction ofclinical and biochemical characteristics of Pompe's disease as discussedabove. Treatment is administered soon after birth, or antenatally if theparents are known to bear variant alpha glucosidase alleles placingtheir progeny at risk.

[0076] Patients with the late onset adult form of Pompe's disease maynot experience symptoms within the first two decades of life. In thisclinical subtype, predominantly skeletal muscles are involved withpredilection of those of the limb girdle, the trunk and the diaphragm.Difficulty in climbing stairs is often the initial complaint. Therespiratory impairment varies considerably. It can dominate the clinicalpicture, or it is not experienced by the patient until late in life.Most such patients die because of respiratory insufficiency. In patientswith the juvenile subtype, symptoms usually become apparent in the firstdecade of life. As in adult Pompe's disease, skeletal muscle weakness isthe major problem; cardiomegaly, hepatomegaly, and macroglossia can beseen, but are rare. In many cases, nightly ventilatory support isultimately needed. Pulmonary infections in combination with wasting ofthe respiratory muscles are life threatening and mostly become fatalbefore the third decade. Treatment with the present methods prolongs thelife of patients with late onset juvenile or adult Pompe's disease up toa normal life span. Treatment also eliminates or significantly reducesclinical and biochemical symptoms of disease.

[0077] Other Uses

[0078] Lysosomal proteins produced in the milk of transgenic animalshave a number of other uses. For example, α-glucosidase, in common withother α-amylases, is an important tool in production of starch, beer andpharmaceuticals. See Vihinen & Mantsala, Crit. Rev. Biochem. Mol. Biol.24, 329-401 (1989) (incorporated by reference in its entirety for allpurpose). Lysosomal proteins are also useful for producing laboratorychemicals or food products. For example, acid α-glucosidase degrades 1,4and 1,6α-glucidic bonds and can be used for the degradation of variouscarbohydrates containing these bonds, such as maltose, isomaltose,starch and glycogen, to yield glucose. Acid α-glucosidase is also usefulfor administration to patients with an intestinal maltase or isomaltasedeficiency. Symptoms otherwise resulting from the presence of undigestedmaltose are avoided. In such applications, the enzyme can beadministered without prior fractionation from milk, as a food productderived from such milk (e.g., ice cream or cheese) or as apharmaceutical composition. Purified recombinant lysosomal enzymes arealso useful for inclusion as controls in diagnostic kits for assay ofunknown quantities of such enzymes in tissue samples.

EXAMPLES Example 1 Construction of Transgenes

[0079] (a) cDNA construct

[0080] Construction of an expression vector containing cDNA encodinghuman acid α-glucosidase started with the plasmid p16,8hlf3 (see DeBoeret al. (1991) & (1993), supra) This plasmid includes bovine αS1-caseinregulatory sequences. The lactoferrin cDNA insert of the parent plasmidwas exchanged for the human acid α-glucosidase cDNA (Hoefsloot et al.EMBO J. 7,1697-1704 (1988)) at the ClaI site and SalI site of theexpression cassette as shown in FIG. 1. To obtain the compatiblerestriction sites at the ends of the α-glucosidase cDNA fragment,plasmid pSHAG2 (id.) containing the complete cDNA encoding humanα-glucosidase was digested with EcoRI and SphI and the 3.3 kbcDNA-fragment was subcloned in pKUN7ΔC a pKUN1 derivative (Konings etal., Gene 46, 269-276 (1986)), with a destroyed ClaI site within thevector nucleotide sequences and with a newly designed polylinker:HindIII ClaI EcoRI SphI XhoI EcoRI SfiI SfiI/SmaI NotIEcoRI*(*=destroyed site). From the resulting plasmid pαgluCESX, the3.3-kb cDNA-fragment could be excised by ClaI and XhoI. This fragmentwas inserted into the expression cassette shown in FIG. 1 at the ClaIsite and XhoI-compatible SalI site. As a result, the expression plasmidp16,8αglu consists of the cDNA sequence encoding human acidα-glucosidase flanked by bovine αS1-casein sequences as shown in FIG. 1.The 27.3-kb fragment containing the complete expression cassette can beexcised by cleavage with NotI (see FIG. 1).

[0081] (b) Genomic Constructs

[0082] Construct c8αgluex1 contains the human acid α-glucosidase gene(Hoefsloot et al., Biochem. J. 272, 493-497 (1990)); starting in exon 1just downstream of its transcription initiation site (see FIG. 2, panelA). Therefore, the construct encodes almost a complete 5′ UTR of thehuman acid α-glucosidase gene. This fragment was fused to the promotersequences of the bovine αS1-casein gene. The αS1-casein initiation siteis present 22 bp upstream of the αS1-casein/acid α-glucosidase junction.The construct has the human acid α-glucosidase polyadenylation signaland 3′ flanking sequences. Construct c8αgluex2 contains the bovineαS1-casein promoter immediately fused to the translation initiation sitein exon 2 of the human acid α-glucosidase gene (see FIG. 2, panel B).Thus, the αS1-casein transcription initiation site and the α-glucosidasetranslation initiation site are 22-bp apart in this construct. Thereforeno α-glucosidase 5′ UTR is preserved. This construct also contains thehuman acid α-glucosidase polyadenylation signal and 3′ flankingsequences as shown in FIG. 2, panel B.

[0083] Construct c8,8αgluex2-20 differs from construct c8αgluex2 only inthe 3′ region. A SphI site in exon 20 was used to fuse the bovineαS1-casein 3′ sequence to the human acid α-glucosidase construct. Thepolyadenylation signal is located in this 3′ αS1-casein sequence (FIG.2, panel C).

[0084] Construct c8,8αgluex2-20 differs from construct c8αgluex2 only inthe 3′ region. A SphI site in exon 20 was used to fuse the bovineαS1-casein 3′ sequence to the human acid α-glucosidase construct. Thepolyadenylation signal is located in this 3′ αS1-casein sequence (FIG.2, panel C).

[0085] Methods for Construction of Genomic Constructs

[0086] Three contiguous BglII fragments containing the human acidα-glucosidase gene were isolated by Hoefsloot et al., supra. Thesefragments were ligated in the BglII-site of pKUN8ΔC, a pKUN7ΔCderivative with a customized polylinker: HindIII ClaI StuI SstI BglIIPvnI NcoI EcoRI SphI XhoI EcoRI* SmaI/SfiI NotI EcoRI* (*=destroyedsite). This ligation resulted in two orientations of the fragmentsgenerating plasmids p7.3αgluBES, p7.3αgluBSE, p8.5αgluBSE, p8.5αgluBES,p10αgluBSE and p10αgluBES.

[0087] Because unique NotI-sites at the ends of the expression cassetteare used subsequently for the isolation of the fragments used formicroinjection, the intragenic NotI site in intron 17 of human acidα-glucosidase had to be destroyed. Therefore, p10αgluBES was digestedwith ClaI and XhoI; the fragment containing the 3′ α-glucosidase end wasisolated. This fragment was inserted in the ClaI and XhoI sites ofpKUN10ΔC, resulting in p10αgluΔNV. Previously pKUN10ΔC (i.e., aderivative of pKUN8ΔC) was obtained by digesting pKUN8ΔC with NotI,filling in the sticky ends with Klenow and subsequently, annealing theplasmid by blunt-ended ligation. Finally, p10αgluΔNV was digested withNotI. These sticky ends were also filled with Klenow and the fragmentwas ligated, resulting in plasmid p10αgluΔNotI.

[0088] Construction of c8αgluex1

[0089] Since the SstI site in first exon of the α-glucosidase gene waschosen for the fusion to the bovine αS1-casein sequence, p8.5αgluBSE wasdigested with BglII followed by a partial digestion with SstI. Thefragment containing exon 1-3 was isolated and ligated into the BglII andSstI sites of pKUN8ΔC. The resulting plasmid was named: p5′αgluex1 (seeFIG. 3, panel A).

[0090] The next step (FIG. 3, panel B) was the ligation of the 3′ partto p5′αgluex1. First, p10αgluΔN was digested with BglII and BamHI. Thisfragment containing exon 16-20 was isolated. Second, p5′αgluex1 wasdigested with BglII and to prevent self-ligation, and treated withphosphorylase (BAP) to dephosphorylate the sticky BglII ends. SinceBamHI sticky ends are compatible with the BglII sticky ends, these endsligate to each other. The resulting plasmid, i.e., p5′3′αgluex1, wasselected. This plasmid has a unique BglII site available for the finalconstruction step (see FIG. 3, panels B and C).

[0091] The middle part of the α-glucosidase gene was inserted into thelatter construct. For this step, p7.3αgluBSE was digested with BglII,the 8.5-kb fragment was isolated and ligated to the BglII digested anddephosphorylated p5′3′αgluex1 plasmid. The resulting plasmid is pαgluex1(FIG. 3, panel C).

[0092] The bovine αS1-casein promoter sequences were incorporated in thenext step via a ligation involving three fragments. The pWE15 cosmidvector was digested with NotI and dephosphorylated. The bovineαS1-casein promoter was isolated as an 8 Rb NotI-ClaI fragment (see deBoer et al., 1991, supra). The human acid α-glucosidase fragment wasisolated from pαgluex1 using the same enzymes. These three fragmentswere ligated and packaged using the Stratagene GigapackII kit in 1046 E.coli host cells. The resulting cosmid c8αgluex1 was propagated in E.coli strain DH5α. The vector was linearized with NotI beforemicroinjection.

[0093] Construction of c8αgluex2 and c8,8αgluex2-20

[0094] The construction of the other two expression plasmids (FIG. 2,panels B and C) followed a similar strategy to that of c8αgluex1. Theplasmid p5′αgluStuI was derived from p8,5αgluBSE by digestion of theplasmid with StuI, followed by self-ligation of the isolated fragmentcontaining exon 2-3 plus the vector sequences. Plasmid p5′αgluStuI wasdigested with PglII followed by a partial digestion of the linearfragment with NcoI resulting in several fragments. The 2.4 kb fragment,containing exon 2 and 3, was isolated and ligated into the NcoI andBglII sites of vector pKUN12ΔC, resulting in p5′αgluex2. Note thatpKUN12ΔC is a derivative of pKUN8ΔC containing the polylinker: ClaI NcoIBglII HindIII EcoRI SphI XhoI SmaI/SfiI NotI.

[0095] The plasmid p10αgluΔNotI was digested with BglII and HindIII. Thefragment containing exons 16-20 was isolated and ligated in p5′αgluex2digested with BglIII and HindIII. The resulting plasmid wasp5′3′αgluex2. The middle fragment in p5′3′αgluex2 was inserted as forpαgluex1. For this, p7.3αglu was digested with BglII. The fragment wasisolated and ligated to the BglII-digested and dephosphorylatedp5′3′αgluex2. The resulting plasmid, pαgluex2, was used in constructionof c8αgluex-20 and c8,8αgluex2-20 (FIG. 2, panels B and C).

[0096] For the construction of third expression plasmid c8,8α gluex2-20(FIG. 2, panel C) the 3′ flanking region of α-glucosidase was deleted.To achieve this, pαgluex2 was digested with SphI. The fragmentcontaining exon 2-20 was isolated and self-ligated resulting inpαgluex2-20. Subsequently, the fragment containing the 3′ flankingregion of bovine αs1-casein gene was isolated from p16,8αglu bydigestion with SphI and NotI. This fragment was inserted intopαgluex2-20 using the SphI site and the NotI site in the polylinkersequence resulting in pαgluex2-20-3αS1.

[0097] The final step in generating c8,8αgluex2-20 was the ligation ofthree fragments as in the final step in the construction leading toc8αgluex1. Since the ClaI site in pαgluex2-20-3′ αS1 and pαgluex2appeared to be uncleavable due to methylation, the plasmids had to bepropagated in the E. coli DAM(−) strain ECO343. The pαgluex2-20-3′ αS1isolated from that strain was digested with ClaI and NotI. The fragmentcontaining exons 2-20 plus the 3′ αS1-casein flanking region waspurified from the vector sequences. This fragment, an 8 kb NotI-ClaIfragment containing the bovine αs1 promoter (see DeBoer (1991) & (1993),supra) and NotI-digested, dephosphorylated pWE15 were ligated andpackaged. The resulting cosmid is c8,8αgluex2-20.

[0098] Cosmid c8αgluex2 (FIG. 2, panel B) was constructed via a coupleof different steps. First, cosmid c8,8αgluex2-20 was digested with SalIand NotI. The 10.5-kb fragment containing the αS1-promoter and the exons2-6 part of the acid α-glucosidase gene was isolated. Second, plasmidpαgluex2 was digested with SalI and NotI to obtain the fragmentcontaining the 3′ part of the acid α-glucosidase gene. Finally, thecosmid vector pWE15 was digested with NotI and dephosphorylated. Thesethree fragments were ligated and packaged. The resulting cosmid isc8αgluex2.

Example 2 Transgenesis

[0099] The cDNA and genomic constructs were linearized with NotI andinjected in the pronucleus of fertilized mouse oocytes which were thenimplanted in the uterus of pseudopregnant mouse foster mothers. Theoffspring were analyzed for the insertion of the human α-glucosidasecDNA or genomic DNA gene construct by Southern blotting of DNA isolatedfrom clipped tails. Transgenic mice were selected and bred.

[0100] The genomic constructs linearized with NotI were also injectedinto the pronucleus of fertilized rabbit oocytes, which were implantedin the uterus of pseudopregnant rabbit foster mothers. The offspringwere analyzed for the insertion of the alpha-glucosidase DNA by Southernblotting. Transgenic rabbits were selected and bred.

Example 3 Analysis of Acid α-Glucosidase in the Milk of Transgenic Mice

[0101] Milk from transgenic mice and nontransgenic controls was analyzedby Western Blotting. The probe was mouse antibody specific for humanacid α-glucosidase (i.e., does not bind to the mouse enzyme). Transgenes1672 and 1673 showed expression of human acid α-glucosidase in milk(FIG. 4). Major and minor bands at 100-110 kD and 76 kD were observed asexpected for α-glucosidase. In milk from non-transgenic mice, no bandswere observed.

[0102] The activity of human acid α-glucosidase was measured with theartificial substrate 4-methylumbelliferyl-α-D-glucopyranoside in themilk of transgenic mouse lines (See Galiaard, Genetic Metabolic Disease,Early Diagnosis and Prenatal Analysis, Elsevier/North Holland,Amsterdam, pp. 809-827 (1980)). Mice containing the cDNA construct(FIG. 1) varied from 0.2 to 2 μmol/ml per hr. The mouse lines containingthe genomic construct (FIG. 2, panel A) expressed at levels from 10 to610 μmol/ml per hr. These figures are equivalent to a production of 1.3to 11.3 mg/l (cDNA construct) and 0.05 to 3.3 g/l (genomic construct)based on an estimated specific activity of the recombinant enzyme of 180μmol/mg (Van der Ploeg et al., J. Neurol. 235:392-396 (1988)).

[0103] The recombinant acid α-glucosidase was isolated from the milk oftransgenic mice, by sequential chromatography of milk on ConA-Sepharose™and Sephadex™ G200. 7 ml milk was diluted to 10 ml with 3 ml Con Abuffer consisting of 10 mM sodium phosphate, pH 6.6 and 100 mM NaCl. A1:1 suspension of Con A sepharose in Con A buffer was then added and themilk was left overnight at 4° C. with gentle shaking. The Con Asepharose beads were then collected by centrifugation and washed 5 timeswith Con A buffer, 3 times with Con A buffer containing 1 M NaCl insteadof 100 mM, once with Con A buffer containing 0.5 M NaCl instead of 100mM and then eluted batchwise with Con A buffer containing 0.5 M NaCl and0.1 M methyl-α-D-mannopyranoside. The acid α-glucosidase activity in theeluted samples was measured using the artificial4-methyl-umbelliferyl-α-D-glycopyranoside substrate (see above).Fractions containing acid α-glucosidase activity were pooled,concentrated and dialyzed against Sephadex buffer consisting of 20 mM Naacetate, pH 4.5 and 25 mM NaCl, and applied to a Sephadex™ 200 column.This column was run with the same buffer, and fractions were assayed foracid α-glucosidase activity and protein content. Fractions rich in acidα-glucosidase activity and practically free of other proteins werepooled and concentrated. The method as described is essentially the sameas the one published by Reuser et al., Exp. Cell Res. 155:178-179(1984). Several modifications of the method are possible regarding theexact composition and pH of the buffer systems and the choice ofpurification steps in number and in column material.

[0104] Acid α-glucosidase purified from the milk was then tested forphosphorylation by administrating the enzyme to cultured fibroblastsfrom patients with GSD II (deficient in endogenous acid α-glucosidase).In this test mannose 6-phosphate containing proteins are bound bymannose 6-phosphate receptors on the cell surface of fibroblasts and aresubsequently internalized. The binding is inhibited by free mannose6-phosphate (Reuser et al., Exp. Cell Res. 155:178-189 (1984)). In atypical test for the phosphorylation of acid α-glucosidase isolated frommilk of transgenic mice, the acid α-glucosidase was added to 104-106fibroblasts in 500 μl culture medium (Ham F10, supplied with 10% fetalcalf serum and 3 mM Pipes) in an amount sufficient to metabolize 1 μmole4-methyl-umbelliferyl-α-D-glucopyranoside per hour for a time period of20 hours. The experiment was performed with or without 5 mM mannose6-phosphate as a competitor, essentially as described by Reuser et al.,supra (1984). Under these conditions acid α-glucosidase of the patientfibroblasts was restored to the level measured in fibroblasts fromhealthy individuals. The restoration of the endogenous acidα-glucosidase activity by acid α-glucosidase isolated from mouse milkwas as efficient as restoration by acid α-glucosidase purified frombovine testis, human urine and medium of transfected CHO cells.Restoration by α-glucosidase form milk was inhibited by 5 mM mannose6-phosphate as observed for α-glucosidase from other sources. (Reuser etal., supra; Van der Ploeg et al., (1988), supra; Van der Ploeg et al.,Ped. Res. 24:90-94 (1988).

[0105] As a further demonstration of the authenticity of α-glucosidaseproduced in the milk, the N-terminal amino acid sequence of therecombinant α-glucosidase produced in the milk of mice was shown to bethe same as that of α-glucosidase precursor from human urine aspublished by Hoefsloot et al., EMBO J. 7:1697-1704 (1988) which startswith AHPGRP.

Example 4 Animal Trial of Alpha-Glucosidase

[0106] Recently, a knock-out mouse model for Pompe's disease has becomeavailable (25) This model was generated by targeted disruption of themurine alpha-glucosidase gene. Glycogen-containing lysosomes aredetected soon after birth in liver, heart and skeletal muscle. Overtclinical symptoms only become apparent at relatively late age (>9months), but the heart is typically enlarged and the electrocardiogramis abnormal.

[0107] Experiments have been carried out using the knock-out (KO) mousemodel in order to study the in vivo effect of AGLU purified fromtransgenic rabbit milk. The recombinant enzyme in these experiments waspurified from milk of the transgenic rabbits essentially as describedabove for purification from transgenic mice.

[0108] 1. Short Term Studies in KO Mouse Model

[0109] Single or multiple injections with a 6 day interval wereadministered to KO mice via the tail vein. Two days after the lastenzyme administration the animals were killed, and the organs wereperfused with phosphate buffered saline (PBS). Tissue homogenates weremade for GLU enzyme activity assays and tissue glycogen content, andultrathin sections of various organs were made to visualize accumulation(via electron microscopy) lysosomal glycogen content. Also thelocalization of internalized AGLU was determined using rabbit polyclonalantibodies against human placenta mature α-glucosidase.

[0110] The results showed that single doses of 0.7 and 1.7 mg AGLU(experiments C and A respectively) was taken up efficiently in vivo invarious organs of groups of two knock-out mice when injectedintravenously. Experiment A also showed that there were no differencesin the uptake and distribution of AGLU purified from two independentrabbit milk sources.

[0111] Increases in AGLU activity were seen in the organs such as theliver, spleen, heart, and skeletal muscle, but not in the brain. Twodays after a single injection of 1.7 mg AGLU to two KO animals, levelsclose to, or much higher than, the endogenous alpha-glucosidase activitylevels observed in organs of two PBS-injected normal control mice or twoheterozygous KO mice were obtained (experiment A). Of the organs tested,the liver and spleen are, quantitatively, the main organs involved inuptake, but also the heart and pectoral and femoral muscles take upsignificant amounts of enzyme. The absence of a significant increase inbrain tissue suggests that AGLU does not pass the blood-brain barrier.The results are summarized in Table 2. TABLE 2 Tissue Uptake of AGLU andGlycogen Content Following Short Term Treatment in KO Mouse ModelPectoral Femoral Liver Spleen Heart Muscle Muscle Tongue Brain Group ActGlc Act Glc Act Glc Act Glc Act Glc Act Glc Act Glc Experiment A animalstreated with single dose of 1.7 mg AGLU (from 2 sources) treated KO 674— — — 263 — — — 24 — — — 0.8 — mice source 1 410 17 3.1 0.4 treated KO454 — — — 76 — — — 12 — — — 0.8 — mice source 2 604 48 10 0.4 untreatedKO 3.1 — — — 0.2 — — — 0.2 — — — 0.2 — mouse untreated 58 — — — 23 — — —11 — — — 57 — normal mouse 37 17 8.2 57 Experiment B animals treatedwith 4 doses of AGLU (1.0, 2.0, 1.0 and 1.4 mg) 6 days apart treated KO1132 70 — — 24 1259 125 87 — — 89 — 0.4 163 mice (13 weeks 944 13 101082 46 116 35 0.2 163 old) treated KO 3375 23 — — 60 1971 49 90 — — 207— 0.7 374 mice (34 weeks old) untreated KO 2.0 406 — — 0.2 3233 1.0 86 —— 1.0 — 0.2 487 mice (13 and 2.0 147 0.3 1748 1.0 87 1.0 0.2 168 34weeks old) untreated 35 6 — — 8.2 0 6.0 1.0 — — 14 — 18 0 normal mice(34 weeks old) Experiment C animals treated with single dose of 0.7 mgtreated KO 582 — 462 — 46 — — — 5.1 — — — 0.4 — mice 558 313 50 3.6 0.4untreated KO 1.1 — 0.8 — 0.3 — — — 0.2 — — — 0.2 — mice 1.6 0.7 0.3 0.30.2

[0112] When two KO mice were injected 4 times every 6 days (experimentB), a marked decrease of total cellular glycogen was observed in bothheart and liver. No effects were observed in skeletal muscle tissueswith regard to total glycogen. However, in general the uptake of AGLU inthese tissues was lower than in the other tissues tested.

[0113] Transmission electron microscopy of the 4 times injected KO miceindicated a marked decrease in lysosomal glycogen in both liver cellsand heart muscle cells. The effects observed in heart tissue arelocalized since in some areas of the heart no decrease in lysosomalglycogen was observed after these short term administrations.

[0114] Western blot analysis using rabbit polyclonal antibodies againsthuman placenta mature alpha-glucosidase indicated complete processing ofthe injected AGLU towards the mature enzyme in all organs testedstrongly suggesting uptake in target tissues, and lysosomal localizationand processing. No toxic effects were observed in any of the threeexperiments.

[0115] Immunohistochemical staining of AGLU was evident in lysosomes ofhepatocytes using a polyclonal rabbit antibody against humanalpha-glucosidase. The presence of AGLU in heart and skeletal tissues ismore difficult to visualize with this technique, apparently due to thelower uptake.

[0116] 2. Long-Term Experiments with the KO Mouse Model

[0117] In longer term experiments, enzyme was injected in the tail veinof groups of two or three KO mice, once a week for periods of up to 25weeks. The initial dose was 2 mg (68 mg/kg) followed by 0.5 mg (17mg/kg)/mouse for 12 weeks. In two groups of mice, this was followed byeither 4 or 11 additional weeks of treatment of 2 mg/mouse. Injectionsstarted when the mice were 6-7 months of age. At this age, clearhistopathology has developed in the KO model. Two days after the lastenzyme administration the animals were killed, and the organs wereperfused with phosphate buffered saline (PBS). Tissue homogenates weremade for AGLU enzyme activity assays and tissue glycogen content, andsections of various organs were made to visualize (via light microscopy)lysosomal glycogen accumulation.

[0118] The results showed that mice treated 13 weeks with 0.5 mg/mouse(Group A, 3 animals/Group) had an increase of activity in the liver andspleen and decreased levels of glycogen in liver and perhaps in heart.One animal showed increased activity in muscles, although there was nosignificant decrease of glycogen in muscle.

[0119] Mice that were treated 14 weeks with 0.5 mg/mouse followed by 4weeks with 2 mg/mouse (Group B, 3 animals/Group) showed similar resultsto those treated for 13 weeks only, except that an increased activitywas measured in the heart and skeletal muscles and decreases of glycogenlevels were also seen in the spleen.

[0120] Mice that were treated 14 weeks with 0.5 mg/mouse followed by 11weeks with 2 mg/mouse (Group C 2 animals/Group) showed similar resultsto the other two groups except that treated mice showed definitedecreases in glycogen levels in liver, spleen, heart and skeletalmuscle. No activity could be detected, even at the highest dose, in thebrain.

[0121] Results of treated and untreated animals in each Group (Groupmeans) are summarized in Table 3. TABLE 3 Tissue Uptake of AGLU andGlycogen Content Following Long Term Treatment in KO Mouse ModelPectoral Quadriceps Gastrocnemius Liver Spleen Heart Muscle MuscleMuscle Brain Group Act Glc Act Glc Act Glc Act Glc Act Glc Act Glc ActGlc Group A animals treated with 0.5 mg/mouse/week for 13 weeks treated713 2 463 n.d 3 86 9 81 6 40 14 66 — — untreated 2 24 1 n.d. 1 111 1 661 50 1 61 — — Group B animals treated with 0.5 mg/mouse/week for 14weeks, followed by 2 mg/mouse./week for 4 weeks treated 2705 1 1628 0 59288 49 120 30 128 44 132 — — untreated 3 11 31 6 1 472 1 113 1 162 1 142— — Group C animals treated with 0.5 mg/mouse/week for 14 weeks,followed by 2 mg/mouse./week for 11 weeks treated 1762 1 1073 2 66 21199 113 37 18 109 32 1 32 untreated 2 45 1 21 1 729 1 291 0 104 0 224 044

[0122] In addition, a very convincing improvement in thehistopathological condition was observed in Group C mice (treated forthe first 14 weeks at 0.5 mg/mouse, followed by 11 weeks at 2 mg/mouse).Clear reversal of pathology was demonstrated in various tissues, such asheart and pectoralis muscle.

[0123] It has been reported that Pompe's disease does not occur when theresidual lysosomal α-glucosidase activity is >20% of average controlvalue (14). The data obtained with the KO mouse model indicates thatthese levels are very well achievable using recombinant precursorenzyme.

Example 5 Human Clinical Trial

[0124] A single phase I study (AGLU1101-01) has been conducted in 15healthy male volunteers. Doses of AGLU ranged from 25 to 800 mg,administered by intravenous infusion to healthy male adult volunteers.Subjects with a history of allergies and hypersensitivities wereexcluded from the study. The subjects were randomized into dose groupsof 5, and each dose Group received AGLU (4 subjects) or placebo (1subject) at each dose level. All subjects received two doses of studydrug, which were administered two weeks apart. The dosing regimen was asfollows:

[0125] A

[0126] 25 mg: Group 1, treatment period 1

[0127] B

[0128] 50 mg: Group 1, treatment period 2

[0129] C

[0130] 100 mg: Group 2, treatment period 1

[0131] D

[0132] 200 mg: Group 3, treatment period 1

[0133] E

[0134] 400 mg: Group 2, treatment period 2

[0135] F

[0136] 800 mg: Group 3, treatment period 2

[0137] P

[0138] placebo (1 subject per Group and treatment period)

[0139] Subjects were administered AGLU or placebo as an infusion on Day1 of each treatment period. The infusions were administered over a 30minute period and subjects were kept in a semi-recumbent position for atleast 2 hours after cessation of infusion.

[0140] Adverse events were recorded just before the start of theinfusion, at 30 minutes (end of infusion) and at 3, 12, 24, 36 and 48hours thereafter as well as on Days 5 and 8 (first period) and days 5, 8and 15 (second period). Vital signs, ECG and physical examinations werealso monitored regularly throughout the treatment period.

[0141] Blood samples were taken for a standard range of clinicallaboratory tests and pharmacokinetics analysis. The subject's urine wascollected and a standard range of laboratory analyses (includingdetermination of AGLU) were performed.

[0142] (a) Laboratory Safety and Adverse Events

[0143] There were no clinically significant changes in laboratoryparameters, clinical signs and ECG measurements in any subjects at anydose Group. The results of adverse event monitoring in all subjects atall doses are summarized in Table 4. TABLE 4 Adverse Event Reports Dose(mg) Adverse Events 25 The reported events were muscle weakness,abnormal vision and fatigue. All events were mild and were deemedunrelated to the test article by the investigator. 50 The reportedevents were headache, rhinitis, nose bleed and paresthesia. All eventswere mild and were deemed unrelated or remotely related to the testarticle by the investigator, except the paresthesia which was classed asmoderate and was deemed possibly related to the test article. 100 Thereported events were rhinitis, headache, fatigue, hematoma and injectionsite reaction. All events were classed as mild. The cases of hematoma,injection site reaction and intermittent headache were deemed possiblyor probably related to the test article by the investigator. The otherevents were deemed to be unrelated. 200 The reported events were nausea,headache, dizziness, fatigue, rhinitis, photophobia, visionabnormalities and euphoria. All events were classed as mild or moderatein intensity. Seven events (including cases of dizziness, nausea andabnormal vision) were deemed to be possibly or probably related to thetest article. 400 The reported events were fatigue and paresthesia. Thereport of fatigue was considered unrelated to the test article, and theparesthesia was deemed possibly related. 800 The reported events werenausea, drowsiness, dizziness, increased sweating, asthenia, shiveringand intermittent headache. All events were classed as mild or moderatein intensity. Eight events (including cases of drowsiness, nausea andasthenia) were deemed to be possibly related to the test article.

[0144] A trial of the safety and efficacy of recombinant acidα-glucosidase as enzyme replacement therapy on infantile and juvenilepatients with glycogen storage disease Type II is conducted. Fourinfantile patients and three juvenile patients are recruited. Infantilesare administered a starting dose of 15-20 mg/kg titrated to 40 mg/kg andjuveniles are administered 10 mg/kg. Patients are treated for 24 weeks.

[0145] Patients are evaluated by the following parameters.

[0146] Standard adverse event reporting including suspected adverseevents

[0147] Laboratory parameters including hematology, clinical chemistryand antibody detection.

[0148] α-glucosidase activity in muscle

[0149] Muscle histopathology

[0150] 12-lead ECG

[0151] Clinical condition including neurological examination

[0152] Non-parametric PK parameters

[0153] Life saving interventions

[0154] Infantile patients are evaluated for the following additionalparameters.

[0155] Left posterior ventricular wall thickness and left ventricularmass index

[0156] Neuromotor development

[0157] Survival

[0158] Glycogen content in muscle

[0159] Juvenile patients are evaluated for the following additionalparameters.

[0160] Pulmonary function

[0161] Muscle strength/timed tests and muscle function

[0162] PEDI/Rotterdam 9-item scale

[0163] The same patients are then subject to additional dosages of alphaglucosidase with infantiles receiving 15, 20, 30 or 40 mg/kg andjuveniles: 10 mg/kg for an additional period of 24 weeks and evaluatedby the parameters indicated above.

[0164] A further phase II clinical trial is performed on eight patientsaged <6 months of age within 2 months after diagnosis at a dosage of 40mg/kg. Patients are treated for 24 weeks and evaluated by the followingcriteria:

[0165] Safety parameters Laboratory safety data

[0166] Adverse event recording

[0167] Primary efficacy parameter: survival without life-savinginterventions (i.e. mechanical ventilation >24 hr) 6 months pastdiagnosis in combination with normal or mildly delayed motor function(BSID II).

[0168] Secondary efficacy: Changes in neuromotor development; changes inleft posterior ventricular wall thickness and left ventricular massindex; Changes in skeletal muscle acid α-glucosidase activity andglycogen content.

[0169] Efficacy can be show by a 50% survival at 6 months post-diagnosiswithout life saving interventions in the α-glucosidase group compared to10% survival in the historical control group in combination with a BSIDII classified as normal or mildly delayed.

[0170] A further clinical trial is performed on juvenile patients. Thepatients are aged >1 year and <35 years of age with juvenile onset ofGSD type IIb The patients are administered 10 mg/kg or 20 mg/kg for aperiod of twenty-four weeks treatment. Treatment is monitored by thefollowing parameters. Safety parameters Laboratory safety data Adverseevent recording Primary efficacy Pulmonary function parameters (e.g.FVC, time on ventilator) Muscle strength Secondary efficacy Life-savinginterventions parameters: Quality of life Skeletal muscle acidα-glucosidase activity Quantitative objective 20% relative improvementin primary efficacy parameters over baseline

[0171] All quantitative measurements relating to efficacy are preferablystatistically significant relative to contemporaneous or historicalcontrols, preferably at p<0.05.

Example 6 Pharmaceutical Formulations

[0172] Alpha-glucosidase is formulated as follows: 5 mg/ml □-Glu, 15 mMsodium phosphate, pH 6.5, 2% (w/w) mannitol, and 0.5% (w/w) sucrose. Theabove formulation is filled to a final volume of 10.5 ml into a 20 cctubing vial and lyophilized. For testing, release and clinical use, eachvial is reconstituted with 10.3 ml* of sterile saline (0.9%) forinjection (USP or equivalent.) to yield 10.5 ml of a 5 mg/ml □-Glusolution that may be directly administered or subsequently diluted withsterile saline to a patient specific target dose concentration. The 10.5ml fill (52.5 mg alpha glucosidase total in vial) includes the USPrecommended overage, that allows extraction and delivery (or transfer)of 10 mls (50 mg). The mannitol serves as a suitable bulking agentshortening the lyophilization cycle (relative to sucrose alone). Thesucrose serves as a cryo/lyoprotectant resulting in no significantincrease in aggregation following reconstitution. Reconstitution of themannitol (only) formulations had repeatedly resulted in a slightincrease in aggregation. Following lyophilization, the cake quality wasacceptable and subsequent reconstitution times were significantlyreduced Saline is preferred to HSA/dextrose for infusion solution. Whensaline is used in combination with lyophilization in 2% mannitol/0.5%sucrose the solution has acceptable tonicity for intravenousadministration. The lyophilized vials containing the 2% mannitol/0.5%sucrose formulation were reconstituted with 0.9% sterile saline (forinjection) to yield 5 mg/ml □-Glu.

Example 7 Infusion Schedule

[0173] The solution is administered via the indwelling intravenouscannula. Patients are monitored closely during the infusion period andappropriate clinical intervention are taken in the event of an adverseevent or suspected adverse event. A window of 48 hours is allowed foreach infusion. An infusion schedule in which the rate of infusionincreases with time reduces or eliminates adverse events.

[0174] Infusions for infantiles can be administered according to thefollowing schedule:

[0175] 5 cc/hr for 60 minutes

[0176] 10 cc/hr for 60 minutes

[0177] ≧40 cc/hr for 30 minutes

[0178] ≧80 cc/hr for the remainder of the infusion

[0179] Infusions for juveniles can be administered according to thefollowing schedule:

[0180] 0.5 cc/kg/hr for 60 minutes

[0181] 1 cc/kg/hr for 60 minutes

[0182] 5 cc/kg/hr for 30 minutes

[0183] 7.5 cc/kg hr for the remainder of the infusion

[0184] While the foregoing invention has been described in some detailfor purposes of clarity and understanding, it will be clear to oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention. All publications and patent documents cited inthis application are incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication or patentdocument were so individually denoted.

1 3 1 6 PRT Artificial Sequence Description of Artificial SequenceN-terminal of recombinant alpha-glucosidase 1 Ala His Pro Gly Arg Pro 15 2 36 DNA Artificial Sequence Description of Artificial Sequence Figure1 flanking sequence 2 ctcgagtatc gattgaattc atctgtcgac gctacc 36 3 18DNA Artificial Sequence Description of Artificial Sequence Figure 1Flanking sequence 3 gcatgcctcg acggtacc 18

What is claimed is:
 1. A method of treating a patient with Pompe's disease, comprising: administering to the patient a therapeutically effective amount of human acid alpha glucosidase.
 2. The method of claim 1, wherein the patient is administered at least 10 mg/kg body weight per week.
 3. The method of claim 1, wherein the patient is administered at least 60 mg/kg body weight per week.
 4. The method of claim 1, wherein the patient is administered at least 120 mg/kg body weight per week.
 5. The method of any of claims 1-4, wherein the patient is administered a single dosage of alpha-glucosidase per week.
 6. The method of any of claims 1-4, wherein the patient is administered three dosages of alpha-glucosidase per week.
 7. The method of any of claims 1-4, wherein the amount is administered per week for a period of at least 24 weeks.
 8. The method of claim 1, wherein the alpha-glucosidase is administered intravenously.
 9. The method of claim 1, wherein the alpha-glucosidase was produced in milk of a transgenic mammal.
 10. The method of claim 1, wherein the patient has infantile Pompe's disease.
 11. The method of claim 10, wherein the patient survives to be at least one year old.
 12. The method of claim 1, wherein the patient has juvenile Pompe's disease.
 13. The method of claim 1, wherein the patient has adult Pompe's disease.
 14. The method of claim 1, wherein the alpha-glucosidase is predominantly in a 110 kD form.
 15. The method of claim 1, further comprising monitoring a level of human acid alpha glucosidase in the patient.
 16. The method of claim 15, further comprising administering a second dosage of human acid alpha glucosidase if the level of alpha-glucosidase falls below a threshold value in the patient.
 17. The method of claim 1, wherein the human alpha glucosidase is administered intravenously and the rate of administration increases during the period of administration.
 18. The method of claim 17, wherein the rate of administration increases by at least a factor of ten during the period of administration.
 19. The method of claim 17, wherein the rate of administration increases by at least a factor of ten within a period of five hours.
 20. The method of claim 17, wherein the patient is administered a series of at least four dosages, each dosage at a higher strength than the previous dosage.
 21. The method of claim 20, wherein the dosages are a first dosage of 0.03-3 mg/kg/hr, a second dosage of 0.3-12 mg/kg/hr, a third dosage of 1-30 mg/kg/hr and a fourth dosage of 2-60 mg/kg/hr.
 22. The method of claim 21, wherein the dosages are a first dosage of 0.1-1 mg/kg/hr, a second dosage of 1-4 mg/kg/hr, a third dosage of 3-10 mg/kg/hr and a fourth dosage of 6-20 mg/kg/hr.
 23. The method of claim 22, wherein the dosages are a first dosage of 0.25-4 mg/kg/hr, a second dosage of 0.9-1.4 mg/kg/hr, a third dosage of 3.6-5.7 mg/kg/hr and a fourth dosage of 7.2-11.3 mg/kg/hr.
 24. The method of claim 23, wherein the dosages are a first dosage of 0.3 mg/kg/hr, a second dosage of 1 mg/kg/hr, a third dosage of 4 mg/kg/hr and a fourth dosage of 12 mg/kg/hr.
 25. The method of any of claims 20-24, wherein the first, second, third and fourth dosages are each administered for periods of 15 min to 8 hours.
 26. The method of any of claims 20-24, wherein the first, second, third and fourth dosages are administered for periods of 1 hr, 1 hr, 0.5 hr and 3 hr respectively.
 27. A pharmaceutical composition comprising human acid alpha glucosidase, human serum albumin, and a sugar in a physiologically acceptable buffer in sterile form.
 28. The pharmaceutical composition of claim 17 comprising human acid alpha glucosidase, human serum albumin, and glucose in sodium phosphate buffer.
 29. A pharmaceutical composition comprising alpha glucosidase, mannitol and sucrose in an aqueous solution.
 30. The pharmaceutical composition of claim 27, wherein the sugar comprises mannitol and sucrose and the concentration of mannitol is 1-3% w/w of the aqueous solution and the concentration of sucrose is 0.1 to 1% w/w of the aqueous solution.
 31. The pharmaceutical composition of claim 27, wherein the concentration of mannitol is 2% w/w and the concentration of sucrose is 0.5% w/w.
 32. A lyophilized composition produced by lyophilizing a pharmaceutical composition comprising human acid glucosidase, mannitol and sucrose in aqueous solution.
 33. A pharmaceutical composition prepared by lyophilizing a first composition comprising human acid alpha-glucosidase, mannitol, sucrose and an aqueous solution to produce a second composition; and reconstituting the lyophilized composition in saline to produce a third composition.
 34. The pharmaceutical composition of claim 33, wherein the human acid alpha-glucosidase is at 5 mg/ml in both the first and third composition, the mannitol is at 2 mg/ml in the first composition, the sucrose is at 0.5 mg/ml in the first composition, and the saline used in the reconstituting step is 0.9% w/w. 